Papillomaviruses are the causative agents of cutaneous and genital warts. Infection with certain subtypes of genital papillomaviruses is the primary risk factor for cervical cancer. Currently there is no preventative vaccine and no consistently effective antiviral therapy. Development of improved treatments will require greater understanding of the properties of these viruses and their interactions with the host cell. The viral E1 protein, an origin-binding helicase essential for viral replication, is an attractive target for potential therapeutic intervention as inhibition of its function should prevent viral replication and propagation. The long-term goal of this research is to understand the structure, function, and regulation of the E1 protein, both as a eukaryotic DNA replication model and as a therapeutic target. Recent results indicate that E1 is covalently modified by addition of a SUMO-1 moiety. Sumoylation is a relatively newly discovered cellular modification system that can affect the stability, intracellular localization, and/or activity of its target substrates. E1 is sumoylated at a single lysine residue, and in the absence of sumoylation at this site E1 replication function is lost because E1 fails to accumulate in the nucleus. These results indicate that sumoylation has an important regulatory function that controls E1 nuclear localization and possibly modulates other E1 activities as well. Recenty, PIAS proteins (Protein Inhibitors of Activated Stat) were shown to SUMO ligases for some substrates, and we have shown the PIAS1 binds E1 and stimulates transient replication. Whether or not this stimulation of replication is the result of PIAS ligase activity enhancing E1 sumoylation or some other function of PIAS1 is unknown. The specific goals of this proposal are to ascertain the mechanism by which sumoylation controls nuclear accumulation, determine the consequences of sumoylation on E1 biochemical activities, and characterize the effect of PIAS proteins on papillomavirus replication and E1 sumoylation. These studies will elucidate a novel cellular regulatory mechanism for E1 function, will define the basic nuclear transport pathway(s) for E1, and will also provide new information about the general role that sumoylation plays in host cell nucleocytoplasmic transport.